FIG. 6.

The p85α mutant inhibits BCR/ABL-mediated leukemogenesis. (A) The p85α mutant inhibits PI-3k activation and phosphorylation of Akt in vivo. 32Dcl3 parental and BCR/ABL-positive counterparts were infected with p85mutFlag-IRES-GFP (p85mut) or empty IRES-GFP retroviral construct (e). PI-3k enzymatic activity was measured in the kinase assay in vitro using phosphatidylinositol as a substrate. Akt activation was assessed by Western analysis detecting the phosphorylated form of Akt; total Akt protein and actin were detected as loading controls. Anti-Flag antibody was used to confirm the expression of p85mut-Flag protein. (B, C) BCR/ABL-expressing 32Dcl3 cells were infected with p85αmutFlag-IRES-GFP (black bars) or empty IRES-GFP (gray bars) retroviral construct. The proliferation potential of GFP⁺ cells was analyzed by trypan exclusion in the presence of bovine serum albumin, FBS, or FBS plus IL-3 (B) or clonogenic assay (C). (D) Mouse mononuclear bone marrow cells were coinfected with pSRα-BCR/ABL and p85mutFlag-IRES-GFP retroviral construct or IRES-GFP empty construct. The clonogenic ability of GFP⁺ cells was evaluated in the absence of growth factors. Results in B, C, and D represent means ± standard deviations from three to four independent experiments (P value calculated by Student's t test). % of clonogenic cells represents the percentage of cells plated that formed colonies. (E) Survival of SCID mice injected with cells transfected with BCR/ABL and empty plasmid (BCR/ABL+E), or BCR/ABL and p85mut (BCR/ABL+p85mut). Survival of the animals was monitored weekly.